The present invention relates to recombinant DNA which encodes the PleI restriction endonuclease (endonuclease) as well as the PleI methyltransferase and the BstNBII methyltransferase (methylase). The present invention also relates to the expression of PleI restriction endonuclease and BstNBII methylase in E. coli cells containing the recombinant DNA.
PleI endonuclease and methyltransferase are found in the strain of Pseudomonas lemoignei (New England Biolabs"" strain collection #418). The endonuclease (R.PleI) recognizes the double-stranded DNA sequence 5xe2x80x2GAGTC 3xe2x80x2 and cleaves DNA 4 and 5 bases downstream generating a one-base 5xe2x80x2 overhanging end. The PleI methyltransferase (M.PleI) recognizes the double-stranded DNA sequence 5xe2x80x2GASTC 3xe2x80x2 and modifies the N6-adenine by addition of a methyl group to become N6-methyladenine in the DNA sequence.
BstNBII methylase (M.BstNBI) is found in the strain of Bacillus stearothermophilus 33M (New England Biolabs"" strain collection #928). It recognizes the double-stranded DNA sequence 5xe2x80x2GASTC 3xe2x80x2 and modifies the N6-adenine by addition of a methyl group to become N6-methyladenine in the DNA sequence. PleI/BstNBII sites that are N6mA modified by M.BstNBII are resistant to both BstNBII and PleI restriction.
Type II and type IIs restriction endonucleases are classes of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) along the DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313, (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2TTT/AAA3xe2x80x2, 5xe2x80x2PuG/GNCCPy3xe2x80x2 and 5xe2x80x2CACNNN/GTG3xe2x80x2 respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2G/AATTC3xe2x80x2.
A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II and a few type IIs restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enable them to resist infection by bacteriophage, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985); Tsp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421 (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225 (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119 (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241 (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,525; Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that sometimes positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer the DNA site resistant to restriction digestion. For example, Dcm methylase modification of 5xe2x80x2CCWGG3xe2x80x2 (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpM methylase can modify the CG dinucloetide and make the NotI site (5xe2x80x2GCGGCCGC3xe2x80x2) refractory to NotI digestion (New England Biolabs"" catalog, 2000-01, page 220). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such over-expression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning the PleI restriction endonuclease from Pseudomonas lemoignei into E. coli by methylase selection and inverse PCR amplification of the adjacent DNA. A methylase gene with high homology to amino-methyltransferases (N6-adenine methylases) was found in the Pseudomonas lemoignei DNA library after methylase selection. This gene was named PleI methylase gene (pleIM).
In order to clone the PleI endonuclease gene in a large DNA fragment, partial ApoI genomic DNA fragment libraries were constructed using the pUC19 vector. Methylase positive clones were obtained. However, no endonuclease activity was detected in any of the M.PleI positive clones.
Since methylase selection failed to yield a PleI endonuclease clone, inverse PCR was employed to amplify the adjacent downstream DNA sequence. An open reading frame was found adjacent to the pleIM gene. This ORF was named pleIr and was expressed along with pleIM in the pUC19 vector. The amount of PleI produced by this clone was virtually undetectable.
To overexpress the pleIM gene, the gene was amplified by PCR and cloned into expression vectors pUC19, pNEB193 and pUC19. None of these constructs conferred 100% resistance to PleI digestion and were therefore not useful for overexpression of the PleI endonuclease.
Methylase selection on the Bacillus stearothermophilus 33M genomic DNA had yielded a N6-adenine methylase that conferred protection against PleI digestion when expressed in the pSXY20 plasmid. This construct was used in conjunction with a pleIR-pAGR3 plasmid in E.coli strain ER2502 and overexpression of pleIR was achieved. Approximately 100,000 units were produced per gram cells.